The era of tumor immunology began with experiments by Prehn and Main, who showed that antigens on the methylcholanthrene (MCA)-induced sarcomas were tumor specific in that transplantation assays could not detect these antigens in normal tissue of the mice (Prehn et al., J. Natl. Cancer Inst. 1 (1957), 769-778). This notion was confirmed by further experiments demonstrating that tumor specific resistance against MCA-induced tumors can be elicited in the autochthonous host, that is, the mouse in which the tumor originated (Klein et al., Cancer Res. 20 (1960), 1561-1572).
Radiation is frequently used in cancer therapy, either as a single regimen or in combination with cytostatic drugs as radiochemotherapy. However, irradiation-resistant tumor clones are limiting the therapeutic efficiency. High cytoplasmic Hsp70 levels have been found to protect tumor cells from apoptotic cell death induced by stress stimuli or exogeneous compounds (Doong et al., Cancer Lett. 188 (2002, 25-32).
Heat shock proteins (Hsps) are highly conserved molecules mediating protection against lethal damage following various stress stimuli in prokaryotic and eukaryotic cells. Also under physiological conditions they support folding of non-native or misfolded proteins and prevent aggregation during proliferation and cellular differentiation (Hartl and Hayer-Hartl, Science 295 (2002), 1852-1858). The best characterized group of chaperones belong to the Hsp70 family. Like other stress proteins, Hsp70s are most efficient if they operate in concert with co-factors as cellular chaperone machineries. Together with J domain co-chaperones (i.e. Hsp40), they support protein folding and assist translocation across membranes (Pilon and Schekman, Cell 97 (1999), 679-682). ATP hydrolysis, stabilizing Hsp70-substrate complexes, is much faster in collaboration with the Hsp-interacting protein Hip (Frydman and Hohfeld, Trends Biochem. Sci. 22 (1997), 87-92). Apoptosis initiated either by exogenous factors (i.e. tumor necrosis factor α, TNFα), or spontaneous cross-linking of the death domain receptors (TNFR1, DR3) is blockable in the presence of the constitutively expressed Hsc70 and the stress-inducible Hsp70 (Jaattela, Exp. Cell Res. 248 (1999), 30-43), in concert with members of the anti-apoptotic Bcl-2-associated athanogene (Bag) family. The human members of this family are Bag-1, -2, -3, -4, -5, and 6. All of them share a highly conserved 45aa BAG domain consisting of three-helix bundles of variable length and a diverse N-terminal sequence (Takayama et al., J. Biol. Chem. 274 (1999), 781-786). Four Bag proteins (Bag-1, -3, -4, -6) have been reported to compete with Hip for binding to the ATPase domain of Hsp70s, and thus promote chaperone activity (Takayama and Reed, Nat. Cell Biol. 3 (2001), 237-241; Doong et al., Cancer Lett. 188 (2002), 25-32). It was also assumed that Bag proteins operate as cellular adaptors targeting Hsp70/Bag complexes to the cytosolic domain of the 55 kDa TNFR1, and thereby inhibit receptor aggregation and activation of the death domains via TRADD, FADD, TRAF, and RIP (Tschopp et al., Curr. Biol. 9 (1999), 381-384). Overexpression of Bag-4, also termed as the silencer of death domain (SODD) has been found to suppress TNF-induced apoptosis on the one hand (Miki and Eddy, Mol. Cell. Biol. 22 (2002), 2536-2543). On the other hand, increased Hsp70 levels confer protection against TNF-mediated lethal shock in mice (Van Molle et al., Immunity 16 (2002), 685-695). Recently, Bag proteins were discussed as important co-factors, affecting the ATPase cycle of Hsp70s (Miki and Eddy, Mol. Cell. Biol. 22 (2002), 2536-2543). The availability of hydrolyzable ATP regulates Bag binding. Apart from TNFR1, Bag proteins interact with Bcl-2, Raf-kinase, androgen-, HGF-, and PDGF receptors. Following stress, when Hsp70 levels are upregulated the Bag/receptor complexes could be replaced by Bag/Hsp70 complexes.
Although the molecular basis for the interaction between the ATPase domain of Hsp70 and the short BAG domain of Bag-4 had been elucidated in detail (Briknarova et al., Nat. Struct. Biol. 8 (2001), 349-372; Sondermann et al., Science 291 (2001), 1553-1557), knowledge on stress-induced modulations within the plasma membrane, where apoptosis might be initiated, are limited. Previously, a tumor-selective Hsp70 plasma membrane localization was demonstrated by cell surface iodination followed by SDS-PAGE and by flow cytometry of viable tumor cells, with intact plasma membrane, using an Hsp70-specific monoclonal antibody (Multhoff et al., Int. J. Cancer 61 (1995), 272-279; Ferrarini et al., Int. J. Cancer 51 (1992), 613-619). These findings are in line with recently published data (Shin et al., J. Biol. Chem. 278 (2003), 7607-7616), who uncovered an abundance of Hsp, including Hsp70, in the extracellular localized part of the plasma membrane of tumor cells by proteomic analysis of surface-bound proteins (Shin et al., J. Biol. Chem. 278 (2003), 7607-7616). Furthermore, it was found that the amount of membrane-bound Hsp70 could be modulated by treatment of tumor cells with membrane-interactive reagents (Botzler et al., Exp. Hematol. 27 (1999), 470-478) or cytostatic drugs (Gehrmann et al., J. Biol. Chem. 383 (2002), 1715-1725). Also γ-irradiation has been found to affect Hsp70 expression levels in tumor cells (Matsumoto et al., Cancer Lett. 92 (1995), 127-133; Sierra-Rivera et al., Radiat. Res. 135 (1993), 40-45; Suzuki and Watanabe, Biochem. Biophys. Res. Commun. 186 (1992), 1257-1264). Since radiation is frequently used in cancer therapy (Hennequin and Favaudon, Eur. J. Cancer 38 (2002), 223-230; Bartelink et al., Eur. J. Cancer 38 (2002), 216-222), there is medical need to understand irradiation-induced effects on the induction of apoptosis and potential mechanisms of resistance (Travis, Acta Oncol. 41 (2002), 323-333; Shinomiya, J. Cell Med. 5 (2001), 240-253).